1. Field of the Invention
The present invention relates to cellulase preparations and compositions having increased or decreased cellulolytic capacity. The invention further relates to a nucleotide sequence of the bg11 gene encoding extracellular .beta.-glucosidase from a filamentous fungi, a plasmid vector containing the gene encoding extracellular .beta.-glucosidase and transformant strains with increased copy numbers of the .beta.-glucosidase (bg11) gene introduced into the genome. More particularly, the present invention relates to Trichoderma reesei strains that have increased or no levels of expression of the bg11 gene resulting in enhanced or no extracellular .beta.-glucosidase protein levels that can be used in conjunction with other compositions to produce a cellulase product having increased or decreased cellulolytic capacity.
2. State of the Art
Cellulases are known in the art as enzymes that hydrolyze cellulose (.beta.-1,4-glucan linkages), thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. As noted by Wood et al., "Methods in Enzymology", 160, 25, pages 234 et seq. (1988) and elsewhere, cellulase produced by a given microorganism is comprised of several different enzyme classes including those identified as exocello-biohydrolases (EC 3.2.1.91) ("CBH"), endoglucanases (EC 3.2.1.4) ("EG"), .beta.-glucosidases (EC 3.2.1.21) ("BG"). Moreover, the fungal classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs and EGs have been isolated from a variety of bacterial and fungal sources including Trichoderma reesei which contains 2 CBHs, i.e., CBH I and CBH II, and at least 3 EGs, i.e., EG I, EG II, and EG III components.
The complete cellulase system comprising components from each of the CBH, EG, and BG classifications is required to efficiently convert crystalline forms of cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose. Moreover, a synergistic relationship is observed between the cellulase components particularly if they are of different classifications. That is to say, the effectiveness of the complete cellulase system is significantly greater than the sum of the contributions from the isolated components of the same classification. In this regard, it is known in the art that the EG components and CBH components synergistic- ally interact to more efficiently degrade cellulose. See, for example, Wood, Biochem. Soc. Trans., 13, pp. 407-410 (1985).
The substrate specificity and mode of action of the different cellulase components varies with classification, which may account for the synergy of the combined components. For example, the current accepted mode of cellulase action is that endoglucanase components hydrolyze internal .beta.-1,4-glucosidic bonds, particularly, in regions of low crystallinity of the cellulose and exo-cellobiohydrolase components hydrolyze cellobiose from the non-reducing end of cellulose. The action of endoglucanase components greatly facilitates the action of exo-cellobiohydrolases by creating new chain ends which are recognized by exo-cellobiohydrolase components.
.beta.-Glucosidases are essential components of the cellulase system and are important in the complete enzymatic breakdown of cellulose to glucose. The .beta.-glucosidase enzymes can catalyze the hydrolysis of alkyl and/or aryl .beta.-D-glucosides such as methyl .beta.-D-glucoside and p-nitrophenyl glucoside, as well as glycosides containing only carbohydrate residues, such as cellobiose. The catalysis of cellobiose by .beta.-glucosidase is important because it produces glucose for the microorganism and further because the accumulation of cellobiose inhibits cellobiohydrolases and endoglucanases thus reducing the rate of hydrolysis of cellulose to glucose.
Since .beta.-glucosidases can catalyze the hydrolysis of a number of different substrates, the use of this enzyme in a variety of different applications is possible. For instance, some .beta.-glucosidases can be used to liberate aroma in fruit by catalyzing various glucosides present therein. Similarly, some .beta.-glucosidases can hydrolyze grape monoterpenyl .beta.-glucosidase which upon hydrolysis, represents an important potential source of aroma to wine as described by Gunata et al, "Hydrolysis of Grape Monoterpenyl .beta.-D-Glucosides by Various .beta.-Glucosidases", J. Agric. Food Chem., Vol. 38, pp. 1232-1236 (1990).
Furthermore, cellulases can be used in conjunction with yeasts to degrade biomass to ethanol wherein the cellulose degrades cellobiose to glucose that yeasts can further ferment into ethanol. This production of ethanol from readily available sources of cellulose can provide a stable, renewable fuel source. The use of ethanol as a fuel has many advantages compared to petroleum fuel products such as a reduction in urban air pollution, smog, and ozone levels, thus enhancing the environment. Moreover, ethanol as a fuel source would reduce the reliance on foreign oil imports and petrochemical supplies.
But the major rate limiting step to ethanol production from biomass is the insufficient amount of .beta.-glucosidase in the system to efficiently convert cellobiose to glucose. Therefore, a cellulase composition that contains an enhanced amount of .beta.-glucosidase would be useful in ethanol production. contrarily, in some cases, it is desirable to produce a cellulase composition which is deficient in, and preferably free of .beta.-glucosidase. Such compositions would be advantageous in the production of cellobiose and other cellooligosaccharides.
.beta.-glucosidases are present in a variety of prokaryotic organisms, as well as eukaryotic organisms. The gene encoding .beta.-glucosidase has been cloned from several prokaryotic organisms and the gene is able to direct the synthesis of detectable amounts of protein in E. coli without requiring extensive genetic engineering, although, in some cases, coupling with a promotor provided by the vector is required. However, .beta.-glucosidases are not produced by such organisms in commercially feasible amounts.
Furthermore, such prokaryotic genes often cannot be expressed and detected after transformation of the eukaryotic host. Thus, in order to use fungal strains, fungal genes would have to be cloned using methods described herein or by detection with the T. reesei bg11 gene by nucleic acid hybridization.
The contribution and biochemistry of the .beta.-glucosidase component in cellulose hydrolysis is complicated by the apparent multiplicity of enzyme forms associated with T. reesei and other fungal sources (Enari et al, "Purification of Trichoderma reesei and Aspergillus niger .beta.-glucosidase", J. Appl. Biochem., Vol. 3, pp. 157-163 (1981); Umile et al, "A constitutive, plasma membrane bound .beta.-glucosidase in Trichoderma reesei", FEMS Microbiology Letters, Vol. 34, pp. 291-295 (1986); Jackson et al, "Purification and partial characterization of an extracellular .beta.-glucosidase of Trichoderma reesei using cathodic run, polyacrylamide gel electrophoresis", Biotechnol. Bioeng., Vol. 32, pp. 903-909 (1988)). These and many other authors report .beta.-glucosidase enzymes ranging in size from 70-80 Kd and in pI from 7.5-8.5. More recent data suggests that the extracellular and cell wall associated forms of .beta.-glucosidase are the same enzyme (Hofer et al, "A monoclonal antibody against the alkaline extracellular .beta.-glucosidase from Trichoderma reesei: reactivity with other Trichoderma .beta.-glucosidases", Biochim. Biophys. Acta, Vol. 992, pp. 298-306 (1989); Messner and Kubicek, "Evidence for a single, specific .beta.-glucosidase in cell walls from Trichoderma reesei QM9414", Enzyme Microb. Technol., Vol. 12, pp. 685-690 (1990)) and that the variation in size and pI is a result of post translational modification and heterogeneous methods of enzyme purification. It is unknown whether the intracellular .beta.-glucosidase species with a pI of 4.4 and an apparent molecular weight of 98,000 is a novel .beta.-glucosidase (Inglin et al, "Partial purification and characterization of a new intracellular .beta.-glucosidase of Trichoderma reesei", Biochem. J., Vol. 185, pp. 515-519 (1980)) or a proteolytic fragment of the alkaline extracellular .beta.-glucosidase associated with another protein (Hofer et al, supra).
Since a major part of the detectable .beta.-glucosidase activity remains bound to the cell wall (Kubicek, "Release of carboxymethylcellulase and .beta.-glucosidase from cell walls of Trichoderma reesei", Eur. J. Appl. Biotechnol., Vol. 13, pp. 226-231 (1981); Messner and Kubicek, supra; Messner et al, "Isolation of a .beta.-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei", Arch. Microbiol., Vol. 154, pp. 150-155 (1990)), commercial preparations of cellulase are thought to be reduced in their ability to produce glucose because of relatively low concentrations of .beta.-glucosidase in the purified cellulase preparation.
To overcome the problem of .beta.-glucosidase being rate limiting in the production of glucose from cellulose using cellulase produced by a filamentous fungi, the art discloses supplementation of the cellulolytic system of Trichoderma reesei with the .beta.-glucosidase of Aspergillus and the results indicate an increase in rate of saccharification of cellulose to glucose. Duff, Biotechnol Letters, 7, 185 (1985). Culturing conditions of the fungi have also been altered to increase .beta.-glucosidase activity in Trichoderma reesei as illustrated in Sternberg et al, Can. J. Microbiol., 23, 139 (1977) and Tangnu et al, Biotechnol. Bioeng., 23, 1837 (1981), and mutant strains obtained by ultraviolet mutation have been reported to enhance the production of .beta.-glucosidase in Trichoderma reesei. Although these aforementioned methods increase the amount of .beta.-glucosidase in Trichoderma reesei, the methods lack practicality and, in many instances, are not commercially feasible.
A genetically engineered strain of Trichoderma reesei or other filamentous fungi that produces an increased amount of .beta.-glucosidase would be ideal, not only to produce an efficient cellulase system, but to further use the increased levels of expression of the ball gene to produce a cellulase product that has increased cellulolytic capacity. Such a strain can be feasibly produced using transformation.
But, in order to transform mutant strains of Trichoderma reesei or other filamentous fungi, the amino acid sequence of the ball gene of Trichoderma reesei or the other filamentous fungi must be first characterized so that the bg11 gene can be cloned and introduced into mutant strains of Trichoderma reesei or other filamentous fungi.
Additionally, once the bg11 gene has been identified, information within linear fragments of the ball gene can be used to prepare strains of Trichoderma reesei and other filamentous fungi which produce cellulase compositions free of .beta.-glucosidase.
Accordingly, this invention is directed, in part, to the characterization of the bg11 gene that encodes for extracellular or cell wall bound .beta.-glucosidase from Trichoderma reesei and other filamentous fungi. This invention is further directed to the cloning of the bg11 gene into a plasmid vector that can be used in the transformation process, and to introduce the ball gene into the Trichoderma reesei or other filamentous fungi genome in multiple copies, thereby generating transformed strains which produce a cellulase composition having a significant increase in .beta.-glucosidase activity. Moreover, cellulase compositions that contain increased cellulolytic capacity are also disclosed.
This invention is further directed, in part, to the deletion or disruption of the ball gene from the Trichoderma reesei or other filamentous fungi genome. In addition, altered copies of the bg11 gene which may change the properties of the enzyme can be reintroduced back into the Trichoderma reesei or other filamentous fungi genome.